The proposed studies address specific hypotheses concerning the molecular and physical chemical mechanisms of sequence-specific DNA recognition by a transcriptional regulatory protein, transcription factor IIIA (TFIIIA). In addition, approaches for analyzing the interactions of TFIIIA with other proteins that form part of the transcription complex are proposed. As with other central cellular processes, such as protein synthesis and processing of pre-mRNAs, transcription occurs in the context of a large macromolecular complex that is assembled on a nucleic acid substrate containing specific signals which direct the production of a functional complex. Being able to describe the structures of the constituent components of these complexes, the processes (such as binding, dissociation, conformational change, and catalysis) leading to their assembly, function, and disassembly, and the relationships between structure and function will be necessary if a fundamental understanding of these central cellular functions is to be achieved. The proposed studies are motivated by this overall goal. TFIIIA is responsible for nucleating the formation of a large macromolecular complex on 5S rRNA genes that is responsible for synthesis of 5S rRNA. Recent structural studies have suggested a novel mechanism of sequence- specific DNA binding involving a dynamic DNA-protein interface in the TFIIIA-5S rRNA gene complex. The validity of this model will be tested by experiments proposed in this application. Previous studies have also suggested the existence of a large energetic strain in the TFIIIA-5S rRNA gene complex, resulting in a "compensatory" mode of binding, and specific experimental tests of a mechanism for producing such energetic strain are proposed. A powerful genetic assay for analyzing the Xenopus TFIIIA-5S rRNA gene interaction will be used to identify mutant forms of TFIIIA that specifically recognize variant 5S rRNA genes, and these will be used both to better understand the specific contacts that occur between the DNA and the protein in the TFIIIA-5S rRNA gene complex as well as reagents for analyzing the function of mutant forms of TFIIIA in vivo. Finally, methods and reagents for analyzing the interaction of TFIIIA-5S rRNA gene complexes with other components of the transcriptional machinery will be developed and used to study these higher order interactions.